Collybistin and gephyrin are novel components of the eukaryotic translation initiation factor 3 complex

Abstract Background Collybistin (CB), a neuron-specific guanine nucleotide exchange factor, has been implicated in targeting gephyrin-GABAA receptors clusters to inhibitory postsynaptic sites. However, little is known about additional CB partners and functions. Findings Here, we identified the p40 subunit of the eukaryotic translation initiation factor 3 (eIF3H) as a novel binding partner of CB, documenting the interaction in yeast, non-neuronal cell lines, and the brain. In addition, we demonstrated that gephyrin also interacts with eIF3H in non-neuronal cells and forms a complex with eIF3 in the brain. Conclusions Together, our results suggest, for the first time, that CB and gephyrin associate with the translation initiation machinery, and lend further support to the previous evidence that gephyrin may act as a regulator of synaptic protein synthesis.

Systematics of the Oswaldoi Complex (Anopheles, Nyssorhynchus) in South America

Abstract Background Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex. Methods DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. Results Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Crystal Engineering of bright luminescent copper iodide clusters with phosphorus and nitrogen-based ligands

Copper(I) halide clusters are recently considered as good candidate for optoelectronic devices such as OLEDs . Although the copper halide clusters, in particular copper iodide, are very well known since the beginning of the 20th century, only in the late ‘70s the interest on these compounds grew dramatically due their particular photophysical behaviour. These complexes are characterized by a dual triplet emission bands, named Cluster Centred (3CC) and Halogen-to-Ligand charge transfer (3XLCT), the intensities of which are strictly related with the temperature. The CC transition, due to the presence of a metallophylic interactions, is prevalent at ambient temperature while the XLCT transition, located preferentially on the ligand part, became more prominent at low temperature. Since these pioneering works, it was easy to understand the photophysical properties of this compounds became more interesting in solid-state respect to solution with an improvement in emission efficiency. In this work we aim to characterize in SS organocopper(I)iodide compounds to valuate the correlation between the molecular crystal structure and the photophysical properties. It is also considered to hike new strategies to synthesize CuI complexes from the wet reactions to the more green solvent free methods. The advantages in using these strategies are evident but, obtain a single crystal suitable for SCXRD analysis from these batches is quite impossible. The structure solution still remains the key point in this research so we tackle this problem solving the structure by X-ray powder diffraction data. When the sample was fully characterized we moved to design and development of the associated OLED-device. Since copper iodide complexes are often insoluble in organic solvents, the high vacuum deposition technique is preferred. A new non-conventional deposition process have also been proposed to avoid the low complex stability in this practice with an in-situ complex formation in a layer-by layer deposition route.

EPR spectroscopy as a tool for the elucidation of non-covalent structuring principles in complex soft matter

This thesis aims at connecting structural and functional changes of complex soft matter systems due to external stimuli with non-covalent molecular interaction profiles. It addresses the problem of elucidating non-covalent forces as structuring principle of mainly polymer-based systems in solution. The structuring principles of a wide variety of complex soft matter types are analyzed. In many cases this is done by exploring conformational changes upon the exertion of external stimuli. The central question throughout this thesis is how a certain non-covalent interaction profile leads to solution condition-dependent structuring of a polymeric system.rnTo answer this question, electron paramagnetic resonance (EPR) spectroscopy is chosen as the main experimental method for the investigation of the structure principles of polymers. With EPR one detects only the local surroundings or environments of molecules that carry an unpaired electron. Non-covalent forces are normally effective on length scales of a few nanometers and below. Thus, EPR is excellently suited for their investigations. It allows for detection of interactions on length scales ranging from approx. 0.1 nm up to 10 nm. However, restriction to only one experimental technique likely leads to only incomplete pictures of complex systems. Therefore, the presented studies are frequently augmented with further experimental and computational methods in order to yield more comprehensive descriptions of the systems chosen for investigation.rnElectrostatic correlation effects in non-covalent interaction profiles as structuring principles in colloid-like ionic clusters and DNA condensation are investigated first. Building on this it is shown how electrostatic structuring principles can be combined with hydrophobic ones, at the example of host-guest interactions in so-called dendronized polymers (denpols).rnSubsequently, the focus is shifted from electrostatics in dendronized polymers to thermoresponsive alkylene oxide-based materials, whose structuring principles are based on hydrogen bonds and counteracting hydrophobic interactions. The collapse mechanism in dependence of hydrophilic-hydrophobic balance and topology of these polymers is elucidated. Complementarily the temperature-dependent phase behavior of elastin-like polypeptides (ELPs) is investigated. ELPs are the first (and so far only) class of compounds that is shown to feature a first-order inverse phase transition on nanoscopic length scales.rnFinally, this thesis addresses complex biological systems, namely intrinsically disordered proteins (IDPs). It is shown that the conformational space of the IDPs Osteopontin (OPN), a cytokine involved in metastasis of several kinds of cancer, and BASP1 (brain acid soluble protein one), a protein associated with neurite outgrowth, is governed by a subtle interplay between electrostatic forces, hydrophobic interaction, system entropy and hydrogen bonds. Such, IDPs can even sample cooperatively folded structures, which have so far only been associated with globular proteins.

In search of CS2(H2O)n = 1–4 clusters

Gaussian-3 and MP2/aug-cc-pVnZ methods have been used to calculate geometries and thermochemistry of CS2(H2O)n, where n = 1–4. An extensive molecular dynamics search followed by optimization using these two methods located two dimers, six trimers, six tetramers, and two pentamers. The MP2/aug-cc-pVDZ structure matched best with the experimental result for the CS2(H2O) dimer, showing that diffuse functions are necessary to model the interactions found in this complex. For larger CS2(H2O)n clusters, the MP2/aug-cc-pVDZ minima are significantly different from the MP2(full)/6-31G* structures, revealing that the G3 model chemistry is not suitable for investigation of sulfur containing van der Waals complexes. Based on the MP2/aug-cc-pVTZ free energies, the concentration of saturated water in the atmosphere and the average amount of CS2 in the atmosphere, the concentrations of these clusters are predicted to be on the order of 105CS2(H2O) clusters∙cm−3 and 102 CS2(H2O)2 clusters∙cm−3 at 298.15 K. The MP2/aug-cc-pVDZ scaled harmonic and anharmonic frequencies of the most abundant dimer cluster at 298 K are presented, along with the MP2/aug-cc-pVDZ scaled harmonic frequencies for the CS2(H2O)n structures predicted to be present in a low-temperature molecular beam experiment.

Syntheses and characterization of monomeric Mo(VI) complexes with bidentate phosphine oxide ligands and dimeric and tetrameric Mo(V) clusters with benzoic acid and phosphinic acid derivatives, containing MoO2, Mo2O2(μ-O)2 and Mo4O4(μ3-O)4

Mo(VI) oxo complexes have been persistently sought after as epoxidation catalysts. Further, Mo(V) oxo clusters of the form M4(µ3-X)4 (M = transition metal, X = O, S) have been rigorously studied due to their remarkable structures and also their usefulness as models for electronic studies. The syntheses and characterizations of new Mo(VI) and Mo(V) oxo complexes have been described in this dissertation. Two new complexes MoO2Cl2Ph2P(O)CH2COOH and MoO2Cl2Ph2P(O)C6H4tBuS(O) were synthesized from reactions of “MoO2Cl2” with ligands Ph2P(O)CH2COOH and Ph2P(O)C6H4tBuS(O). Tetrameric packing arrangements comprised of hydrogen bonds were obtained for the complex MoO2Cl2Ph2P(O)CH2COOH and the ligand Ph2P(O)CH2COOH. Further the stability of an Mo-O bond was preferred over the Mo-S bond even though this resulted in the formation of a more strained seven membered ring. Tetranuclear Mo(V) complexes of the form [Mo4(µ3-O)4(µ-O2PR2)4O4], (PR2 = PPh2, PMe2) were synthesized using reactions of MoO2(acac)2 with diphenyl and dimethyl phosphinic acids, in ethanol. In the crystal structure of these complexes four Mo=O units are interconnected by four triply bridging oxygen atoms and bridging phosphinate ligands. The complex exhibited fourfold symmetry as evidenced by a single 31P NMR peak for the P atoms in the coordinated ligands. Reaction of WO2(acac)2 with Ph2POOH in methanol resulted in a dimeric W(VI) complex [(CH3O)2(O)W(µ-O)( µ-O2PPh2)2W(O)(CH3O)2] which contained a packing disorder in its crystal structure. Similar reactions of MoO2(acac)2 with benzoic acid derivatives resulted in dimeric complexes of the form [Mo2O2(acac)2(µ-O)(µ-OC2H5)(µ-O2CR)] (R = C6H5, (o-OH)C6H4, (p-Cl)C6H4, (2,4-(OH)2)C6H3, (o-I)C6H4) and one tetrameric complex [Mo2O2(acac)2(µ-O)(µ-OC2H5)(µ-O2C)C6H4(p-µ-O2C)Mo2O2(acac)2(µ-O)(µ-OC2H5)] with terephthalic acid. 1H NMR proved very useful in the prediction of the formation of dimers with the substituted benzoic acids, which were also confirmed by elemental analyses. The reductive capability of ethanol proved instrumental in the syntheses of Mo(V) tetrameric and dimeric clusters. Synthetic details, IR, 1H and 31P NMR spectroscopy and elemental analyses are reported for all new complexes. Further, single crystal X-ray structures of MoO2Cl2Ph2P(O)CH2COOH, MoO2Cl2Ph2P(O)C6H4tBuS(O), [Mo4(µ3-O)4(µ-O2PR2)4O4], (PR2 = PPh2, PMe2), [(CH3O)2(O)W(µ-O)( µ-O2PPh2)2W(O)(CH3O)2] and [Mo2O2(acac)2(µ-O)(µ-OC2H5)(µ-O2CR)] (R = C6H5, (o-OH)C6H4) are also presented.

Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

A Novel Fluorescence-based Genetic Strategy Identifies Mutants of Saccharomyces cerevisiae Defective for Nuclear Pore Complex Assembly

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.

Nuclear Pore Complex Number and Distribution throughout the Saccharomyces cerevisiae Cell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.

Characterization of a Novel Human SMC Heterodimer Homologous to the Schizosaccharomyces pombe Rad18/Spr18 Complex

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27–28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.

Do Clusters Generate Greater Innovation and Growth? An Analysis of European Regions. Bruges European Economic Research (BEER) Papers 21/October 2011

The analysis of clusters has attracted considerable interest over the last few decades. The articulation of clusters into complex networks and systems of innovation -- generally known as regional innovation systems -- has, in particular, been associated with the delivery of greater innovation and growth. However, despite the growing economic and policy relevance of clusters, little systematic research has been conducted into their association with other factors promoting innovation and economic growth. This article addresses this issue by looking at the relationship between innovation and economic growth in 152 regions of Europe during the period between 1995 and 2006. Using an econometric model with a static and a dynamic dimension, the results of the analysis highlight that: a) regional growth through innovation in Europe is fundamentally connected to the presence of an adequate socioeconomic environment and, in particular, to the existence of a well-trained and educated pool of workers; b) the presence of clusters matters for regional growth, but only in combination with a good ‘social filter’, and this association wanes in time; c) more traditional R&D variables have a weak initial connection to economic development, but this connection increases over time and, is, once again, contingent on the existence of adequate socioeconomic conditions.

Targeting a complex transcriptome: The construction of the mouse full-length cDNA encyclopedia

We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large. numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, S'-end clusters identify regions that are potential promoters for 8637 known genes and S'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

Overlapping species boundaries and hybridization within the Monastraea "annularis" reef coral complex in the Pleistocene of the Bahama Islands

Recent molecular analyses indicate that many reef coral species belong to hybridizing species complexes or "syngameons." Such complexes consist of numerous genetically distinct-species or lineages, which periodically split and/or fuse as they extend through time. During splitting and fusion, morphologic intermediates form and species overlap. Here we focus on processes associated with lineage fusion, specifically introgressive hybridization, and the recognition of such hybridization in the fossil record. Our approach involves comparing patterns of ecologic and morphologic overlap in genetically characterized modern species with fossil representatives of the same or closely related species. We similarly consider the long-term consequences of past hybridization on the structure of modern-day species boundaries. Our study involves the species complex Montastraea annularis s.l. and is based in the Bahamas, where, unlike other Caribbean locations, two of the three members of the complex today are not genetically distinct. We measured and collected colonies along linear transects across Pleistocene reef terraces of last interglacial age (approximately 125 Ka) on the islands of San Salvador, Andros, and Great Inagua. We performed quantitative ecologic and morphologic analyses of the fossil data, and compared patterns of overlap among species with data from modern localities where species are and are not genetically distinct. Ecologic and morphologic analyses reveal "moderate" overlap (>10%, but statistically significant differences) and sometimes "high" overlap (no statistically significant differences) among Pleistocene growth forms (= "species"). Ecologic analyses show that three species (massive, column, organ-pipe) co-occurred. Although organ-pipes had higher abundances in patch reef environments, columnar and massive species exhibited broad, completely overlapping distributions and had abundances that were not related to reef environment. For morphometric analyses, we used multivariate discriminant analysis on landmark data and linear measurements. The results show that columnar species overlap "moderately" with organ-pipe and massive species. Comparisons with genetically characterized colonies from Panama show that the Pleistocene Bahamas species have intermediate morphologies, and that the observed "moderate" overlap differs from the morphologic separation among the three modern species. In contrast, massive and columnar species from the Pleistocene of the Dominican Republic comprise distinct morphologic clusters, similar to the modern species; organ-pipe species exhibit "low" overlap (